Decontamination of Mycotoxin-Contaminated Feedstuffs and Compound Feed
5. Feed Additives for the Prevention of Mycotoxin Effects
Feed additives are mixed with contaminated diet to minimize the effect of mycotoxins on the animal prior to intake or during digestion [44,45].
The use of feed additives or supplements that decrease animal exposure to mycotoxins can be viewed as a means of enhancing animal welfare. These feed supplements are referred to as the substances blended into feed (e.g., mineral clay, micro-organism, yeast cell wall), adsorbing or detoxifying mycotoxins in the digestive tract of animals (biological detoxification) [46]. These additives have received increasing attention from the feed industry and numerous products have been developed and some of them have already been tested on animals and marketed.
European Regulation (EC) No 1831/2003 of 22 September 2003 on animal feed additives has been revised and the category of technological feed additives includes a special functional group [47]. That is a group which is described as “substances that can suppress or decrease the absorption of food through mycotoxins, encourage the excretion of mycotoxins or alter their mode of action”, under
Commission Regulation (EC) No 386/2009 of 12 May 2009 [48]. It should be pointed out that the use of such products does not mean that the animal feed exceeding the established maximum limits may be used. Their use should rather improve the quality of the feed which is lawfully on the market, providing an additional guarantee for the protection of animal and public health. Therefore, after adding an additive, these additives may not be used as compatible in non-conforming camouflage consignments. Following a request for technical assistance, in July 2010, the European Food Safety Authority (EFSA) through its Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) issued a statement where it detailed the additional information that would be required to perform an assessment of safety and efficacy of this new group of additives [49]. This statement lists only the requirements which are not common in relation to the rest of technological additives. In 2012, EFSA published several guidelines on its website pertaining to the marketing of several feed additives and safety measures [50]. This guidance document follows the structure and definitions of Regulation (EC) No 1831/2003 and it is intended to assist the applicant in the preparation and the presentation of its application, as foreseen in Article 7.6 of Regulation (EC) No 1831/2003 [48].
Various materials have been tested as mycotoxin-detoxifying agents in order to avoid deleterious impacts of mycotoxins on livestock (mainly poultry and swine). They work either in adsorption or in the bonding or transformation of mycotoxins to their surfaces (biotransformation), depending on their mode of action. Biotransformation of mycotoxins can be caused by the addition of enzymes or micro-organisms generating such enzymes [46].
Mycotoxin binders are nutritionally inert adsorbents that reduce mycotoxin absorption from the gastrointestinal tract by integrating them into contaminated feed, thereby preventing and decreasing mycotoxicosis and transportation of mycotoxins into animal products [46]. The adsorbent materials are designed to behave like a “chemical sponge”, preventing the blood and target organ absorption and later distribution of mycotoxins. The effectiveness of adsorbent on mycotoxin seems to depend on the chemical structure. The main feature is the physical adsorbent structure, i.e., the total distribution of load and load, the dimensions of pores and the available surface. On the other side, adsorbing mycotoxins also have a major part to play, such as polarity, solubility, form and load distribution.
The stability of the sorbent toxin bond and the efficacy over a broad pH range are important criteria for the assessment of possible mycotoxin binders because a product has to be implemented on the entire gastrointestinal tract [51]. The feed composition can also have a major impact on adsorption effectiveness [51]. Potential absorbent materials include activated carbon, aluminosilicates (bentonite, zeolite, phyllosilicates, etc.), complex indigestible carbohydrates (cellulose, polysaccharides in the cell walls of yeast and bacteria such as glucomannans, peptidoglycans, and others), and synthetic polymers such as cholestyramine and polyvinylpyrrolidone and derivatives [20,45,46,52–54]. Many studies have shown that the formation of stable connections of these adsorbent products has a strong affinity with mycotoxins. These are found in a number of fluid systems, such as beer, wine, milk, and peanut oil.
Activated carbon is a widely used adsorption material that has an outstanding adsorption ability with a wide surface region. It is recommended for multiple digestive toxins as a general toxic adsorbing agent and is frequently suggested (The Merck Veterinary Manual, Eighth Edition, Merck & Co., Inc., Whitehouse Station, NJ) [55]. Activated carbon effectiveness depends on the source materials, the surface area and the distribution of the pores on the adsorption characteristics of the activated carbon.
The surface features of activated carbons are greatly altered by preparation techniques and chemical treatments. The contrasting findings regarding the capacity of activated carbon for mycotoxin binding can explain different adsorbing characteristics of different carbonaceous materials [51]. Activated carbon adsorbs most mycotoxins effectively in water, whereas animals are less or not affected by mycotoxicosis. For aflatoxin B1and ochratoxin A adsorption, the highest capacity for in vitro activated carbon was noted whereas deoxynivalenol adsorption was lower. The efficacy of activated carbon has been demonstrated in vivo and in vitro by vibrant gastrointestinal models for deoxynivalenol, nivalenol, zearalenone, aflatoxin, ochratoxin A, diacetoxyscirpenol and T-2 toxins [44,53,56–60]. Responses to charcoal in cows, broilers, turkey poults, rats and mink suggest that charcoal may not be as effective in
binding aflatoxin as the clay-based binders. The biomarker assay in rats did not confirm the in vivo efficacy of activated carbon to bind fumonisin [52,61]. Also, research conducted with weaning pigs showed that they were not effectively protected against the adverse effects of consuming fumonisin B1
by adding activated carbon to contaminated feed [62]. Finally, although having a potential for acute exposure to a number of mycotoxins, activated carbon is a non-specific sequester with large variability in efficacy, which reduces possibilities for its practical application.
Mycotoxin binders are the largest and most complicated class of silicate minerals [63–67]. There are two major sub-classes in this group, phyllosilicate, and tectosilicate [68,69]. The phyllosilicate sub-class mineral clays include significant adsorbents such as the montmorillonite/smectite group, the kaolinite group and the illite (or clay-mica) group [51]. Montmorillonite is a predominantly layered, oxygen-coordinated, phyllosilicate consisting of octahedral aluminum and tetrahedral silicon layers.
The bentonite is usually impure smectite clay. The tectosilicates include important and highly studied zeolites. Zeolites consist of SiO4and AlO4tetrahedrons having a cage-like structure that is infinite in three dimensions. In such minerals, some tetravalent silicones are replaced by trivalent aluminum, which results in inorganic cations, such as sodium, calcium and potassium ions, that have a lack of a positive charge. Clay minerals, primarily montmorillonite, have been used in the early 1970s to reduce aflatoxin toxicity [70]. There is ample literature on this subject, mainly in the field of in vitro water studies [53,63,64,66,71], and animal feed trials [20,72–74]. The use of smectite in human nutrition was also tested for its safety as well as the efficacy in the decrease of aflatoxin biomarkers [64,75–77].
In Europe, bentonite is allowed as a feed additive for all animal species, as well as for mitigation of mycotoxin contamination for ruminants, swine, and poultry (1m558). It is also used for control of radionuclide contamination and as an anticaking agent (1m558i). The chemisorption of aflatoxin to smectites involves the formation of a complex by theβ-keto-lactone or bilactone system of aflatoxin with uncoordinated metal ions in the mineral. Aflatoxin B1is able to be attached on the surface of the mineral particle and in its interlayers. A huge difference in the effectiveness of bentonites in sequestration of aflatoxin B1was shown in several in vitro studies [51]. These studies indicated that aflatoxin B1
bentonite adsorption efficacy may rely on the physical, chemical and mineralogical characteristics of the smectite, including clay contents, the capacities of the cation exchange (CECs), the interlayer cation hydrate radius, distributions of particle size and the specific surface area. Notwithstanding these findings, an important correlation has not been well created between smectites minero-chemical and physicochemical characteristics and aflatoxin B1adsorption. Therefore, there is still no predictive model of aflatoxin B1adsorption by the bentonite as the crystal-chemical variation in the smectite group is complex. Recently, the study by D’Ascanio et al. [67] showed a strong correlation between aflatoxin adsorption parameters and the geological origin of samples. In adsorbing toxin at distinct pH values, sedimentary bentonites were considerably better than hydrothermal bentonites [51]. The extent of aflatoxin B1-adsorption was negative and linear with the extent of desorption. Mineralogical and physicochemical analyses confirmed that some physical and chemical properties of bentonites correlate linearly with AFB1adsorption. However, these studies cannot be deemed to be conclusive since it is still hard to depict the link between properties of these mineral adsorbents and aflatoxin B1 adsorption/desorption. Due to the complexity of interactions and factors that can affect the adsorption of the aflatoxins by smectites, further research is required to describe the mechanisms of adsorption [51].
However, bentonite cannot be used as a binder for all mycotoxins due to their limited binding effects. Several in vivo studies have previously shown that aluminosilicates do not significantly adsorb other mycotoxins, such as cyclopiazonic acid and ergotamine, zearalenone, deoxynivalenol, T-2 toxin, ochratoxin A and others. The selective chemisorption of bentonites for aflatoxins can be overcome by chemical modifications. These include changes in the surface characteristics, resulting in enhanced hydrophobicity when structural load balancing cations are exchanged with molecular heavyweight amines [66,78]. Several in vitro studies showed the binding efficacy of modified montmorillonite and clinoptilolite against zearalenone and ochratoxin A [20]. Aflatoxin B1was adsorbed with non-modified zeolites. However, the in vivo ineffectiveness of these binders in sequestering a large spectrum of
mycotoxins has been recently observed in piglets [79], and some of those clay forms were pointed out to the potential toxicity [80,81].
Recently, questions have been raised about the nanotechnology solution of mycotoxin risk [82].
One of the most promising methods is the use of carbon-based nanomaterials. Graphene has shown a huge surface and a high mycotoxin binding capacity. Polymeric nanoparticles have also been drawn to attention; they may replace adsorbents or contain a substance that would improve the organism’s health status. Modified nanodiamonds synthesized by detonation were proposed as intestinal adsorbent of aflatoxins [83]. Highly advanced surfaces, and the existence on the surface of nanoparticles of multiple functional chemical-active groups, hydrocarbon fragments, and metal micro impurities, establish their elevated affinity to biomolecular sorption. The findings of in vitro experiments, showing that nanodiamonds adsorb aflatoxin B1from aqueous solutions at different pH, were confirmed by in vivo experiments with rats [83]. In order to confirm the effectivity and safety of this adsorbent on animal species, further studies including well-designed in vitro trials are needed. The practical and economic feasibility aspects should also be taken into account [82].
The formation of bonds between polymers, such as cholestyramine, divinylbenzene-styrene and polyvinylpyrrolidone, and mycotoxins were confirmed in vitro and in vivo [52]. Cholestyramine is a binding resin that has proven to bind bile acids in the gastrointestinal tract and decrease low-density lipoproteins and cholesterol. It has been shown that cholestyramine is an efficient binder for ochratoxin A, fumonisins and zearalenone in vitro [52,57,61]. Efficacy of cholestyramine in the dietary concentration of 2% on top of compound feed was confirmed in experiments with feed contaminated by zearalenone and by the biomarker assay in vivo in laboratory animals for fumonisins [52,57,61].
Cholestyramine efficacy for detoxification of zearalenone was also confirmed during other studies on laboratory animals [84]. A polyvinylpyrrolidone, a synthetic water-soluble polymer, was researched as a binder for mycotoxins as well [85]. It was shown that polyvinylpyrrolidone is able to bind aflatoxin B1and zearalenone, while it did not alleviate the toxicity of deoxynivalenol in pigs. It should be noted that the high costs of polymers are a limiting factor for its practical applications.
The mycotoxin-sequestering capacity of different high-fiber feedstuffs, such as hays (e.g., alfalfa hay) or straws (e.g., wheat straw), was recognized a long time ago, but there are principally practical experiences, e.g., in equine nutrition, without scientific assessment. The positive effect of alfalfa fiber was first proved against zearalenone in laboratory animals and pigs and also against T-2 toxicosis in laboratory animals, respectively [86–89]. However, it should also be mentioned that, besides its positive effects, alfalfa fiber is a potential source ofFusariumcontamination, and its high inclusion rates (15–25%) required in the diet may cause digestive-physiological disturbances. Micronized wheat fiber has recently been found effective in decreasing the accumulation of ochratoxin A in laboratory animals’
liver and kidney tissues. When used at an inclusion level of 20 kg/t, it significantly increased the excretion of ochratoxin A via the feces [90,91]. Recently, Avantaggiato et al. [92] showed that a red-grape pomace (pulp and skin) can sequester distinct mycotoxins quickly and simultaneously. Aflatoxin B1, followed by zearalenone, ochratoxin A and fumonisin B1, was the most affected mycotoxin. In pigs, using a urinary biomarker method the effectiveness of grape pomace in secreting mycotoxins has been confirmed [79]. Aflatoxin B1(67%) and zearalenone (69%) considerably lowered the urinary mycotoxin biomarker of the grape pomace. Taking these outcomes into consideration, the authors indicated that the use of grape pomace as a large-spectrum adsorbent material has its potential. Greco et al. [93]
recorded evidence on the capacity of food plants and by-products other than grape pomace and wheat fibers to absorb mycotoxins. The research results are highly innovative and prove that a wide range of mycotoxins is also available in some dietary fibers. Aflatoxin B1, zearalenone, and ochratoxin A were most of the adsorbed mycotoxins. Adsorption of aflatoxin B1, zearalenone, and ochratoxin A was not impacted by pH, and the adsorbed fraction was not released when acid-to-neutral pH increased.
Mycotoxin fumonisin B1has been adsorbed to a lesser level in this research and its adsorption has been affected by a medium’s pH.
Polymeric humic materials comprise several binding sites and are being incorporated into humans as a compound to minimize bacterial endotoxins absorption and systemic accessibility. A high-quality humic acid derivative, called oxyhumate, has been reported to have the mycotoxin-sequestering capacity and recommended for use against aflatoxicosis based on in vivo studies in chickens [94].
The excellent connection capability of humic substances with zearalenone was an exciting finding, as assessed in vitro research [60]. These compounds should, therefore, be further tested in vivo.
The other groups of fiber components are the cell wall components of the yeastSaccharomyces cerevisiae, the mannan-oligosaccharides (MOS) or their esterified form withβ-D-glucan (esterified glucomannan), which showed the considerable binding ability for several mycotoxins in vivo.
Saccharomyces cerevisiae and lactic acid bacteria are the two most important food fermentation microorganisms that have proven to bind various mycotoxins [20,54,95]. The reversible and strain and dose-dependent phenomenon of mycotoxin binding of some chosen lactic acid bacteria was outlined and did not influence the viability of the lactic acid bacteria. It should be noted that there may be a relationship between lactic acid bacteria and the accumulation of mycotoxins through two particular procedures like binding and biosynthesis inhibition. There could, therefore, be a high value for the reduction of mycotoxin exposure for lactic acid cultures with a strong anti-fungal, anti-mycotoxigenic and mycotoxin potential [96].
Fungal conidia can bind mycotoxin individually or together (between 29 and 60%), particularly zearalenone and ochratoxin A [97]. Saccharomyces cerevisiaelive yeast was shown to reduce the detrimental effects of aflatoxin in broiler diets [20,34,95,98,99]. The aflatoxin protective effect of live yeast was confirmed in rats, but thermolyzed yeast was shown ineffective. The potential to bind several mycotoxins was shown as fibrous material from the cell wall of the yeast. It has been shown that esterified glucomannan polymer obtained from the yeast cell wall separately and in combination binds aflatoxin, ochratoxin and toxin T-2. Additions of 0.5 or 1.0 kg/t doses of esterified glucomannan to aflatoxin-contaminated diets resulted in broiler chicks, with dose-dependent reactions. Similarly, in relation to aflatoxin-contaminated diets of dairy cows, esterified glucan polymer considerably decreased residues of aflatoxin in milk. The esterified glucan polymer may have the capability to bind several mycotoxins. A glucan polymer-bound both T-2 toxin and zearalenone in vitro, and it was protective against depression in antioxidant activities resulting from T-2 toxin consumed by growing quail. A glucan polymer product has protected swine, broilers, and hens against some of the detrimental effects of multiple mycotoxins, while another glucan polymer product did not alleviate the toxic effects on mink consuming diets contaminated with fumonisin B1, ochratoxin A, moniliformin and zearalenone [98,99]. These polysaccharides, in addition to binding mycotoxins, also provide other functions to regulate the damage of mycotoxins in animal organs, including modulation of immune operations and binding gastrointestinal pathogens [100–102]. It should also be noticed that many trials are carried out using commercial products which may not consist exclusively of glucomannans, but contain tiny quantities of aluminosilicates that are specifically added to bind aflatoxins.
Numerous studies and several comprehensive reviews as above demonstrate the increasing interest in the use of mycotoxin-detoxifying agents as technological feed additives.
The best way to evaluate mycotoxin binders is with in vivo experiments. Naturally, in vivo models are perfect and hard to conduct in theory. It is complicated, costly, and time-consuming to collect the definitive data. Individual bioassays with the same strain, age, body weight, and dietary type should take place in vivo research to achieve coherent outcomes. Differences in farm conditions and types, health, development, and maturity of animals may also have an effect on outcomes. Binders with varying rates of incorporation, distinct mycotoxins, animal species, age, gender, and environment should be assessed as well. Moreover, according to the EU Guideline 2001/79/EC on additives for use in animal nutrition [50], the in vivo efficacy of binders should be proven by using an experimental design justified according to the claim for the use of the additive, and by using specific biological markers such as tissue residues or changes in biochemical parameters [103]. The chosen biomarker for exposure should be specific for each mycotoxin and target species, closely related to exposure and
easy to detect with sensitive analytical methods validated for the matrix used [3]. EFSA has proposed different biomarkers for exposure to aflatoxin B1, deoxynivalenol, zearalenone, ochratoxin A, and fumonisins. To date, the majority of the research on the effectiveness of mycotoxin binders has only been done on the feed consumption and measurements of performance. Few in vivo research papers have explored potential side impacts on animal performance or the health of these agents.